Cells go to great lengths to maintain their cytoplasmic water concentration and failure to do so can be disastrous. In the case of kidney cells, which are responsible for maintaining the water balance of the whole organism, dysfunction can mean severe and global metabolic disease. We have in hand unique melanoma cell lines genetically deficient in actin-binding protein (ABP-) which display abnormal volume regulation, motility, and continuously extrude large surface blebs over their entire surface. Genetic rescue of ABP in these cells leads to an ablation of blebbing and restoration of normal motility, surface topology, and volume regulation. These cells, therefore, are ideal to analyze the role of ABP and of the actin cytoskeleton in stabilizing the plasma membrane and in the control of cell volume and blebbing. We will use a molecular biological approach to define which functional domains of the ABP molecule are required for osmotic stability and for ablation of abnormal surface blebbing. ABP truncates of the following construction will be stably transfected into ABP- melanoma cells: (a) lacking the dimer site, but having normal membrane and actin binding sites; (b) without a membrane glycoprotein binding site, but having normal dimerization and actin binding sites; (c) missing actin binding sites but having dimer and glycoprotein binding sites. These maneuvers will dissect whether filament attachment to the membrane (truncate a), filament crosslinking but not membrane binding (truncate b), or membrane binding alone (truncate c) are sufficient for rescue or whether all sites are necessary. Similarly, we will establish the ABP domain(s) necessary for activation of the osmotically-dependent membrane channel activity (K+/C1- channels). Preliminary experiments have revealed that only ABP+- rescued cells open volume regulatory channels. We will also purify and characterize the membrane protein bound to melanoma ABP and define how it interacts with ion channels to affect cell volume.